General processes for producing optically active alcohols with oxidoreductases require coenzymes. In order to avoid complete consumption of the coenzymes in these reactions, efficient regeneration of the coenzymes during the reduction/oxidation reactions is important. Examples of coenzymes required for the production of optically active alcohols include pyridine nucleotide coenzymes such as reduced β-nicotinamide adenine dinucleotide phosphate (NADPH, NADP+ for its oxidized form) and reduced β-nicotinamide adenine dinucleotide (NADH, NAD+ for its oxidized form). One known strategy to regenerate these coenzymes is to use glucose dehydrogenase (GDH), formate dehydrogenase (FDH), or the like.
Although alcohol dehydrogenases with good properties (stability, solvent resistance, oxidation resistance) and specific activity have been known, their coenzyme dependency has not been optimized yet. Therefore, their productivity of optically active alcohols is limited even when NADPH or NADH is regenerated. Development of techniques to optimize the coenzyme dependency of alcohol dehydrogenase according to the coenzyme dependency of a coenzyme regeneration enzyme, that is, to optimize alcohol dehydrogenase to be NADPH- or NADH-dependent, will realize easier optimization of production processes of optically active alcohols and more efficient production of optically active alcohols.
Variant screening to identify an enzyme with altered coenzyme dependency takes a lot of effort. Instead, rational designing has been attempted to design a desired enzyme variant with altered coenzyme dependency based on three-dimensional structure data of the enzyme (Patent Literature 1 and Non Patent Literature 1).
Alcohol dehydrogenases (ADH) are a representative group of enzymes including members whose variants with altered coenzyme dependency are known to be obtainable by rational designing. In particular, recent studies have reported success in altering the coenzyme dependency of the short-chain dehydrogenase/reductase (SDR) family, which has about 250 amino acid residues (Non Patent Literature 2, Non Patent Literature 3). Yet, there is no report of success on the medium-chain dehydrogenase/reductase (MDR) family, which has about 350 amino acid residues.